Deconstructing and recapitulating complex biological processes requires multidimensional control of molecular
function. Optogenetics has emerged as a versatile means of achieving this capability, as demonstrated by the impact
of channelrhodopsins and halorhodopsins on neuroscience. Developmental and regenerative biology would also be
transformed by new optogenetic technologies, as tissue formation requires the coordination of multiple signaling
pathways in space and time. To date, nearly all non-rhodopsin optogenetic systems have relied on proximity-based
mechanisms to control protein function, using natural photoreceptors such as phytochromes, cryptochromes, and light-
oxygen-voltage (LOV) domains. While this methodology has yielded valuable tools, is it not broadly applicable across
the proteome.
Allosteric optical control is another powerful means of regulating protein activity in space and time. However,
relatively few examples of such optogenetic systems have been reported to date, likely due to the challenge of
developing functional photoreceptor-signaling protein chimeras. Our project seeks to address this challenge by
recapitulating how photoreceptors likely evolved in nature: the random insertion of light-sensing domains into signaling
proteins and the selection of photoresponsive variants. In particular, we envision that transposon technologies could
greatly expedite optogenetic engineering by bypassing the bottleneck of evaluating individual photoreceptor-
functionalized constructs. We will employ Tn5 and Tn7 transposase-mediated insertional mutagenesis to create large
random libraries of photoreceptor-signal protein chimeras, and we will identify photoresponsive variants using cell-
based reporters and flow cytometry (Aim 1). We will then apply directed evolution and targeted mutagenesis to optimize
these optogenetic tools and evaluate their efficacy in zebrafish models (Aim 2).
Our proof-of-concept studies focus on LOV domains, taking advantage of their compact size, structural and
functional diversity, and amenability to protein engineering. We will apply these microbial and plant-derived
photoreceptors to Smoothened (SMO) and GLl1, canonical regulators of the Hedgehog signaling pathway, and we
evaluate photoresponsive LOV-SMO and LOV-GLI1 constructs in zebrafish embryos. We anticipate that our
transposon-based strategy will be broadly applicable to functionally diverse proteins, accelerating the development of
new optogenetic tools and expanding the scope of optobiology. Our long-term goal is to assemble an optogenetic
toolbox for manipulating the molecular pathways that regulate tissue patterning and regeneration (e.g., Wnt, BMP, and
Notch signaling), optically intercepting multiple points within each pathway.